Methods and systems for disease detection

Abstract

Provided herein are methods of determining that a subject has or is at risk of having a disease (e.g., cancer) using nucleic acid molecules derived from a cell-free biological sample of the subject.

What is claimed is: 
     
         1 . A method for determining that a subject has or is at risk of having a disease, comprising:
 (a) obtaining a cell-free biological sample of said subject;   (b) using a plurality of nucleic acid molecules derived from said cell-free biological sample to generate a plurality of circular nucleic acid molecules;   (c) subjecting said plurality of circular nucleic acid molecules or derivatives thereof to sequencing to generate a plurality of sequence reads; and   (d) processing said plurality of sequence reads against a plurality of reference sequence reads to identify at least a subset of said plurality sequence reads as corresponding to at least a subset of said plurality of reference sequence reads, thereby determining that said subject has or is at risk of having said disease.   
     
     
         2 . A method for determining that a subject has or is at risk of having a disease, comprising:
 (a) obtaining a cell-free biological sample of said subject;   (b) using a plurality of nucleic acid molecules derived from said cell-free biological sample to generate a plurality of circular nucleic acid molecules;   (c) subjecting said plurality of circular nucleic acid molecules or derivatives thereof to sequencing to identify a plurality of breakpoint sequences; and   (d) processing said plurality of breakpoint sequences against a plurality of reference breakpoint sequences to identify at least a subset of said plurality breakpoint sequences as corresponding to at least a subset of said plurality of reference breakpoint sequences, thereby determining that said subject has or is at risk of having said disease.   
     
     
         3 . The method of  claim 2 , wherein each of said plurality of breakpoint sequences comprises a 5′ end and a 3′ end of a nucleic acid molecule derived from said cell-free biological sample. 
     
     
         4 . The method of  claim 1  or  claim 2 , wherein (b) comprises ligating ends of each of said plurality of nucleic acid molecules to one another. 
     
     
         5 . The method of  claim 1  or  claim 2 , wherein (b) comprises coupling an adapter to a 3′ end, a 5′ end, or both a 5′end and a 3′end of each of said plurality of nucleic acid molecules. 
     
     
         6 . The method of  claim 1  or  claim 2 , further comprising, subsequent to (b), subjecting each of said plurality of circular nucleic acid molecules to nucleic acid amplification to generate a plurality of amplification products of said plurality of circular nucleic acid molecules, wherein (c) comprises subjecting said plurality of amplification products or derivatives thereof to sequencing to generate a plurality of sequence reads or a plurality of breakpoint sequences of said plurality of amplification products or derivatives thereof. 
     
     
         7 . The method of  claim 6 , wherein said nucleic acid amplification is effected by a polymerase having strand-displacement activity. 
     
     
         8 . The method of  claim 6 , wherein said nucleic acid amplification is effected by a polymerase that does not have strand-displacement activity. 
     
     
         9 . The method of  claim 6 , wherein said nucleic acid amplification comprises contacting said circular nucleic acid molecule to an amplification reaction mixture comprising random primers. 
     
     
         10 . The method of  claim 6 , wherein said nucleic acid amplification comprises contacting said circular nucleic acid molecule to an amplification reaction mixture comprising one or more primers, each of which specifically hybridizes to a different target sequence via sequence complementarity. 
     
     
         11 . The method of  claim 1  or  claim 2 , wherein said plurality of nucleic acid molecules is single stranded. 
     
     
         12 . The method of  claim 1  or  claim 2 , wherein said plurality of nucleic acid molecules comprises cell-free DNA or RNA. 
     
     
         13 . The method of  claim 1  or  claim 2 , wherein said plurality of nucleic acid molecules is from a tumor. 
     
     
         14 . The method of  claim 1  or  claim 2 , further comprising processing said sequence to monitor a progression or regression of said disease in said subject in response to treatment. 
     
     
         15 . The method of  claim 1  or  claim 2 , wherein said sequencing comprises a method selected from one or more of sequencing by synthesis, sequencing by ligation, nanopore sequencing, nanoball sequencing, ion detection, sequencing by hybridization, polymerized colony (POLONY) sequencing, nanogrid rolling circle sequencing (ROLONY), and ion torrent sequencing. 
     
     
         16 . The method of  claim 1  or  claim 2 , wherein said cell-free biological sample comprises less than 75 nanograms of nucleic acids. 
     
     
         17 . The method of  claim 1  or  claim 2 , wherein said cell-free biological sample comprises a bodily fluid. 
     
     
         18 . The method of  claim 17 , wherein said bodily fluid is urine, saliva, blood, serum, plasma, tears, sputum, cerebrospinal fluid, synovial fluid, mucus, bile, semen, lymph, amniotic fluid, menstrual fluid, or combinations thereof. 
     
     
         19 . The method of  claim 1  or  claim 2 , further comprising computer processing said sequence to identify an epigenetic modification in said sequence. 
     
     
         20 . The method of  claim 19 , wherein said epigenetic modification is selected from methylation, phosphorylation, ubiquitination, sumoylation, acetylation, ribosylation, citrullination, and fragmentation. 
     
     
         21 . The method of  claim 1  or  2 , wherein said disease is cancer. 
     
     
         22 . The method of  claim 21 , wherein said cancer is selected from the group consisting of colon cancer, non-small cell lung cancer, small cell lung cancer, breast cancer, hepatocellular carcinoma, liver cancer, skin cancer, malignant melanoma, endometrial cancer, esophageal cancer, gastric cancer, ovarian cancer, pancreatic cancer, brain cancer, leukemia, lymphoma, and myeloma. 
     
     
         23 . The method of  claim 1 , further comprising using at least said subset of said plurality sequence reads identified in (d) to output an electronic report indicating that said subject has or is at risk of having said disease. 
     
     
         24 . The method of  claim 2 , further comprising using at least said subset of said plurality breakpoint sequences identified in (d) to output an electronic report indicating that said subject has or is at risk of having said disease. 
     
     
         25 . The method of  claim 1 , further comprising using at least said subset of said plurality sequence reads identified in (d) to provide a therapeutic intervention to said subject for said disease. 
     
     
         26 . The method of  claim 2 , further comprising using at least said subset of said plurality breakpoint sequences identified in (d) to provide a therapeutic intervention to said subject for said disease. 
     
     
         27 . The method of  claim 1 , further comprising using at least said subset of said plurality sequence reads identified in (d) to treat said subject for said disease. 
     
     
         28 . The method of  claim 2 , further comprising using at least said subset of said plurality breakpoint sequences identified in (d) to treat said subject for said disease. 
     
     
         29 . The method of  claim 27  or  28 , wherein said subject is treated by administering a chemotherapy or immunotherapy to said subject. 
     
     
         30 . The method of any one of  claims 23  to  28 , wherein said disease is cancer. 
     
     
         31 . The method of  claim 1 , further comprising using at least said subset of said plurality sequence reads identified in (d) to monitor said subject for a progression or regression of said subject. 
     
     
         32 . The method of  claim 2 , further comprising using at least said subset of said plurality breakpoint sequences identified in (d) to monitor said subject for a progression or regression of said subject. 
     
     
         33 . The method of  claim 1 , wherein (d) comprises determining that at least 0.1% of said plurality of sequence reads correspond to said plurality of reference sequence reads. 
     
     
         34 . The method of  claim 2 , wherein (d) comprises determining that at least 0.1% of said plurality of breakpoint sequences correspond to said plurality of reference breakpoint sequences. 
     
     
         35 . A method for determining that a subject has or is at risk of having a disease, comprising:
 (a) accessing (i) a first database comprising a plurality of sequence reads, wherein a sequence read of said plurality of sequence reads comprises multiple copies of a sequence of a nucleic acid molecule from said subject; and (ii) a second database comprising a plurality of reference sequence reads; and   (b) processing said plurality of sequence reads from said first database against said plurality of reference sequence reads from said second database to identify at least a subset of said plurality sequence reads as corresponding to at least a subset of said plurality of reference sequence reads, thereby determining that said subject has or is at risk of having said disease.   
     
     
         36 . A method for determining that a subject has or is at risk of having a disease, comprising:
 (a) accessing (i) a first database comprising a plurality of breakpoint sequences, wherein a breakpoint sequence of said plurality of breakpoint sequences comprises multiple copies of a sequence of a 5′ end and a 3′ end of a nucleic acid molecule of said subject; and (ii) a second database comprising a plurality of reference breakpoint sequences; and   (b) processing said plurality of breakpoint sequences from said first database against said plurality of reference breakpoint sequences from said second database to identify at least a subset of said plurality breakpoint sequences as corresponding to at least a subset of said plurality of reference breakpoint sequences, thereby determining that said subject has or is at risk of having said disease.   
     
     
         37 . The method of  claim 35  or  claim 36 , wherein said first database and said second database are different databases. 
     
     
         38 . The method of  claim 35  or  claim 36 , wherein said disease is cancer. 
     
     
         39 . A system for determining that a subject has or is at risk of having a disease, comprising:
 (a) a first database comprising a plurality of sequence reads, wherein a sequence read of said plurality of sequence reads comprises multiple copies of a sequence of a nucleic acid molecule of said subject;   (b) a second database comprising a plurality of reference sequence reads; and   (c) one or more computer processors that are individually or collectively programed to process said plurality of sequence reads from said first database against said plurality of reference sequence reads from said second database to identify at least a subset of said plurality sequence reads as corresponding to at least a subset of said plurality of reference sequence reads, thereby determining that said subject has or is at risk of having said disease.   
     
     
         40 . A system for determining that a subject has or is at risk of having a disease, comprising:
 (a) a first database comprising a plurality of breakpoint sequences, wherein a breakpoint sequence of said plurality of breakpoint sequences comprises multiple copies of a sequence of a 5′ end and a 3′ end of a nucleic acid molecule of said subject;   (b) a second database comprising a plurality of reference breakpoint sequences; and   (c) one or more computer processors that are individually or collectively programed to process said plurality of breakpoint sequences from said first database against said plurality of reference breakpoint sequences from said second database to identify at least a subset of said plurality breakpoint sequences as corresponding to at least a subset of said plurality of reference breakpoint sequences, thereby determining that said subject has or is at risk of having said disease.   
     
     
         41 . The system of  claim 39  or  claim 40 , wherein said first database and said second database are different databases. 
     
     
         42 . The system of  claim 39  or  claim 40 , wherein said disease is cancer. 
     
     
         43 . A non-transitory computer-readable medium comprising machine-executable code that, upon execution by one or more computer processors, implements a method for determining that a subject has or is at risk of having a disease, comprising:
 (a) accessing (i) a first database comprising a plurality of sequence reads, wherein a sequence read of said plurality of sequence reads comprises multiple copies of a sequence of a nucleic acid molecule from said subject; and (ii) a second database comprising a plurality of reference sequence reads; and   (b) processing said plurality of sequence reads from said first database against said plurality of reference sequence reads from said second database to identify at least a subset of said plurality sequence reads as corresponding to at least a subset of said plurality of reference sequence reads, thereby determining that said subject has or is at risk of having said disease.   
     
     
         44 . A non-transitory computer-readable medium comprising machine-executable code that, upon execution by one or more computer processors, implements a method for determining that a subject has or is at risk of having a disease, comprising:
 (a) accessing (i) a first database comprising a plurality of breakpoint sequences, wherein a breakpoint sequence of said plurality of breakpoint sequences comprises multiple copies of a sequence of a 5′ end and a 3′ end of a nucleic acid molecule of said subject; and (ii) a second database comprising a plurality of reference breakpoint sequences; and   (b) processing said plurality of breakpoint sequences from said first database against said plurality of reference breakpoint sequences from said second database to identify at least a subset of said plurality breakpoint sequences as corresponding to at least a subset of said plurality of reference breakpoint sequences, thereby determining that said subject has or is at risk of having said disease.   
     
     
         45 . The non-transitory computer-readable medium of  claim 43  or  claim 44 , wherein said first database and said second database are different databases. 
     
     
         46 . The non-transitory computer-readable medium of  claim 43  or  claim 44 , wherein said disease is cancer.